Two-layer Analyte Sensor

ABSTRACT

An analyte sensor and a method for making the analyte sensor are disclosed. In one aspect, the analyte sensor includes a sensing membrane having a crosslinked network with an embedded analyte sensing component. In another aspect, the analyte sensor includes a protective membrane adjacent to the surface of the sensing membrane. The protective membrane can be a crosslinked, hydrophilic copolymer having methacrylate-derived backbone chains of first methacrylate-derived units, second methacrylate-derived units and third methacrylate-derived units. The first and second methacrylate-derived units have hydrophilic side chains, and the third methacrylate-derived units in different backbone chains are connected by hydrophilic crosslinks. A method for making the analyte sensor is also disclosed.

BACKGROUND

Unless otherwise indicated herein, the materials described in thissection are not prior art to the claims in this application and are notadmitted to be prior art by inclusion in this section.

The continuous or semi-continuous monitoring of physiological parametershas applications in many areas of modern medicine. Electrochemical-basedsensors are believed to be particularly suitable for the monitoring andquantification of analytes (e.g., glucose) in bodily fluid samples(e.g., blood, tear film, urine or interstitial fluid samples). The useof an electrochemical-based sensor that employs an analyte sensingcomponent, (e.g., an enzyme) in conjunction with an electrode(s) allowsfor the quantification of an analyte in a liquid sample by detecting theproduct(s) produced from the reaction of the analyte sensing componentand the analyte.

SUMMARY

In one aspect, an analyte sensor is disclosed. The analyte sensorincludes a sensing membrane having a crosslinked network with anembedded analyte sensing component. The cross-linked network can includeone or more proteins that are crosslinked with carbon-nitrogen doublebonds between the nitrogen atoms of amine groups on the proteins andcarbon atoms in crosslinking groups. The sensing membrane can beadjacent to a surface of an electrode.

In another aspect, the analyte sensor includes a protective membraneadjacent to a surface of the sensing membrane. The protective membranecan be a crosslinked, hydrophilic copolymer having methacrylate-derivedbackbone chains of first methacrylate-derived units, secondmethacrylate-derived units and third methacrylate-derived units. Thefirst and second methacrylate-derived units have hydrophilic side chainsthat can be the same or different, and the third methacrylate-derivedunits in different backbone chains are connected by hydrophiliccrosslinks.

In another aspect, methods for forming the analyte sensor is disclosed.The formation of the sensing membrane can involve forming a sensingmixture of one or more proteins, a crosslinking agent, and an analytesensing component, depositing the sensing mixture onto a surface of anelectrode, and curing the deposited sensing mixture to provide a sensingmembrane. The protective membrane can be formed by forming a copolymermixture of an initiator, a first methacrylate monomer, a dimethacrylatemonomer, and a second methacrylate monomer, depositing the copolymermixture on a surface of the sensing membrane, and curing the depositedcopolymer mixture. The first and second methacrylate monomers can havehydrophilic side chains suitable to provide the first and secondmethacrylate-derived units of the protective membrane, respectively.

These as well as other aspects, advantages, and alternatives, willbecome apparent to those of ordinary skill in the art by reading thefollowing detailed description, with reference where appropriate to theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of current produced by six example glucose sensors atglucose concentrations of 50 μM, 200 μM, 400 μM, 700 μM and 1,000 μM inphosphate buffered saline (PBS).

FIG. 2 is a graph of the relationship between current and glucoseconcentration observed in the six example glucose sensors of FIG. 1 atglucose concentrations of 50 μM, 200 μM, 400 μM, 700 μM and 1,000 μM inphosphate buffered saline (PBS).

FIG. 3 is a representative diagram of a crosslinked, hydrophiliccopolymer, in accordance with an example embodiment. The diagram shows atwo-backbone section of the copolymer, and each 1SC (“first side chain”)represents a hydrophilic side chain connected to a firstmethacrylate-derived unit, and each 2SC (“second side chain”) representsa hydrophilic side chain connected to a second methacrylate-derivedunit. Each CROSSLINK represents a hydrophilic crosslink between thirdmethacrylate-derived units in different backbone chains.

DETAILED DESCRIPTION

The following detailed description describes various features andfunctions of the disclosed systems and methods with reference to theaccompanying figures. In the figures, similar symbols typically identifysimilar components, unless context dictates otherwise. The illustrativemethod and system embodiments described herein are not meant to belimiting. It will be readily understood that certain aspects of thedisclosed methods and systems can be arranged and combined in a widevariety of different configurations, all of which are contemplatedherein.

In one aspect, an analyte sensor is disclosed. The analyte sensorincludes: a sensing membrane, wherein the sensing membrane can include:

-   -   a crosslinked network including one or more proteins and        crosslinks, wherein the proteins are crosslinked through        carbon-nitrogen double bonds between amine group nitrogen atoms        on the proteins and carbon atoms in the crosslinks; and    -   an analyte sensing component embedded in the crosslinked        network.

In some embodiments, the analyte sensor is an enzyme-based biosensor.Biosensors can convert a signal produced by ananalyte-concentration-dependent biochemical reaction into a measurablephysical signal, such as an optical or electrical signal. Biosensors canbe used in the detection of analytes in clinical, environmental,agricultural and biotechnological applications. Analytes that can bemeasured in clinical assays of fluids of the human body include, forexample, glucose, lactate, cholesterol, bilirubin, proteins, lipids andelectrolytes. The detection of analytes in biological fluids, such asblood, tear film, or intestinal fluid, can be important in the diagnosisand the monitoring of many diseases.

In some embodiments, the analyte sensor can be a component of abody-mountable device, such as an eye-mountable, tooth-mountable, orskin-mountable device. The eye-mountable device can be configured tomonitor health-related information based on one or more analytesdetected in a tear film (the term “tear film” is used hereininterchangeably with “tears” and “tear fluid”) of a user wearing theeye-mountable device. For example, the eye-mountable device can be inthe form of a contact lens that includes a sensor configured to detectone or more analytes (e.g., glucose). The eye-mountable device can alsobe configured to monitor various other types of health-relatedinformation.

In some embodiments, the body-mountable device may comprise atooth-mountable device. The tooth-mountable device may take the form ofor be similar in form to the eye-mountable device, and be configured todetect at least one analyte in a fluid (e.g., saliva) of a user wearingthe tooth-mountable device.

In some embodiments, the body-mountable device may comprise askin-mountable device. The skin-mountable device may take the form of orbe similar in form to the eye-mountable device, and be configured todetect at least one analyte in a fluid (e.g., perspiration, blood, etc.)of a user wearing the skin-mountable device.

The sensor as described herein can include one or more conductiveelectrodes through which current can flow. In some embodiments, thesensing membrane can be adjacent to a surface of an electrode. Dependingon the application, the electrodes can be configured for differentpurposes. For example, a sensor can include a working electrode, areference electrode, and a counter-electrode. Also possible aretwo-electrode systems, in which the reference electrode serves as acounter-electrode. The working electrode can be connected to thereference electrode via a circuit, such as a potentiostat.

The electrode can be formed from any type of conductive material and canbe patterned by any process that be used for patterning such materials,such as deposition or photolithography, for example. The conductivematerials can be, for example, gold, platinum, palladium, titanium,carbon, copper, silver/silver-chloride, conductors formed from noblematerials, metals, or any combinations of these materials. Othermaterials can also be envisioned.

The sensing membrane can have a cross-linked network of one protein or amixture of one or more different proteins. The proteins of the sensingmembrane can be substantially unreactive in biochemical reactions, whichwill limit interference with the analyte sensing component. In someembodiments, the protein is bovine serum albumin.

The proteins can be about 15% by weight to about 50% by weight of thesensing membrane. In some embodiments, the proteins are about 15% byweight to about 20% by weight, about 20% by weight to about 25% byweight, about 25% by weight to about 30% by weight, about 30% by weightto about 35% by weight, about 35% by weight to about 40% by weight,about 40% by weight to about 45% by weight, or about 45% by weight toabout 50% by weight of the membrane.

The proteins of the sensing membrane are covalently bound throughcrosslinks, forming a crosslinked network. The crosslinks can be betweenthe one or more proteins in the sensing membrane, but can also bebetween the analyte sensing component, and/or one or more proteinsand/or another analyte sensing component. In some embodiments, thecrosslinks can be formed through carbon-nitrogen double bonds betweenthe nitrogen atoms of amine groups on the proteins and/or analytesensing component and carbon atoms in the crosslinks. These crosslinkscan be derived from dialdehhyde compounds. For example, the crosslinksof the sensing membrane can have the structure of formula (I):

where A is independently a protein or an analyte sensing component,R^(a) is C₀-C₄alkyl or a hydrophilic group, and R′ is independentlyhydrogen or —C₁-C₁₂alkyl. The hydrophilic group can be soluble in wateror a water-miscible solvent, such as an alcohol, and have one or moreheteroatoms (e.g., nitrogen, oxygen or sulfur). In some embodiments, thecrosslinks have one or more hydroxy groups. It is understood fromformula (I) that the crosslinks have two carbons in addition to theR^(a) group. Thus, crosslinks referred to herein as having a certainnumber of carbon atoms (e.g., C₄) will have an R^(a) group with two lesscarbon atoms (e.g., C₂). For example, “C₄alkyl crosslinks” have an R^(a)group that is C₂alkyl.

In some embodiments, the crosslinks can be formed through amide bondsbetween the nitrogen atoms of amine groups on the proteins and/oranalyte sensing component and carbonyl groups in the crosslinks. Thesecrosslinks can be derived from di-carbonyl compounds. For example, thecrosslinks of the sensing membrane can have the structure of formula(Ia):

where A and R^(a) are as described in formula (I).

In some embodiments, the crosslinks of the sensing membrane include oneor more alkylene oxide units. The alkylene oxide units can be in theform of a polymer, such as poly(ethylene glycol), poly(propyleneglycol), poly(butylene oxide) or a mixture thereof, and can be acopolymer including a combination of two or three different alkyleneoxide units. In some embodiments, the poly(alkylene oxide) of thecrosslinks is a block copolymer including blocks of two or threedifferent poly(alkylene oxide) polymers. In certain embodiments, thepoly(alkylene oxide) is a block copolymer of poly(ethylene glycol) andpoly(propylene glycol). In other embodiments, the crosslinks and thesecond methacrylate-derived units include poly(ethylene glycol).

In some embodiments, the crosslinks of the sensing membrane include oneor more ethylene oxide units. For example, the crosslinks (e.g., Ra informula (I) above) can have the structure of formula (Ib):

where w is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In certain embodiments, w is an average value of from about 2 to about250.

In other embodiments, w in the crosslinks of formula (Ib) is such thatthe number average molecular weight (M_(n)) of the PEG portion (withinthe brackets in formula (Ib)) of the crosslinks is about 100 to about10,000. For example, w can be selected such that the M_(n) of the PEGportion of the crosslinks falls within a range in Table 1:

TABLE 1 M_(n) range of the PEG portion of the crosslinks in the sensingmembrane (values are approximate). Low High 100 200 200 300 300 400 400500 500 600 600 700 700 800 800 900 900 1,000 1,000 2,000 2,000 3,0003,000 4,000 4,000 5,000 5,000 6,000 7,000 8,000 8,000 9,000 9,000 10,000

In some embodiments, the crosslinks of the sensing membrane areC₂-C₆alkyl. The crosslinks can be derived from oxaldehyde,malonaldehyde, succinaldehyde, glutaraldehyde or adipaldehyde.

The analyte sensing component is embedded, i.e., surrounded by thecrosslinked network of sensing membrane. The embedded analyte sensingcomponent is immobilized and can interact with a corresponding analyteof interest. In some embodiments, the analyte sensing component includesan enzyme.

The analyte sensing component of the analyte sensor can be selected tomonitor physiological levels of a specific analyte. For example,glucose, lactate, cholesterol and various proteins and lipids can befound in body fluids, including, for example, tear film, and can beindicative of medical conditions that can benefit from continuous orsemi-continuous monitoring.

The analyte sensing component can be an enzyme selected to monitor oneor more analytes. For example, physiological cholesterol levels can bemonitored with cholesterol oxidase, lactate levels with lactate oxidase,and glucose levels with glucose oxidase or glucose dehydrogenase (GDH).

In some embodiments, the analyte sensing component can be an enzyme thatundergoes a chemical reaction with an analyte to produce detectablereaction products. For example, a copolymer including glucose oxidase(“GOx”) can be situated around the working electrode to catalyze areaction with glucose to produce hydrogen peroxide (H₂O₂). As shownbelow, the hydrogen peroxide can then be oxidized at the workingelectrode to release electrons to the working electrode, which generatesa current.

The current generated by either reduction or oxidation reactions can beapproximately proportionate to the reaction rate. Further, the reactionrate can be dependent on the rate of analyte molecules reaching theelectrochemical sensor electrodes to fuel the reduction or oxidationreactions, either directly or catalytically through a reagent. In asteady state, where analyte molecules diffuse to the electrochemicalsensor electrodes from a sampled region at approximately the same ratethat additional analyte molecules diffuse to the sampled region fromsurrounding regions, the reaction rate can be approximatelyproportionate to the concentration of the analyte molecules. The currentcan thus provide an indication of the analyte concentration.

In other embodiments, the analyte sensing component is glucosedehydrogenase (GDH). In certain instances, the use of GDH can includethe addition of a cofactor such as flavin adenine dinucleotide (FAD),nicotinamide adenine dinucleotide (NAD), flavin mononucleotide,pyrroloquinoline quinone (PQQ) or a coenzyme.

The analyte sensing component can be present in about 40% to about 80%by weight in the sensing membrane. Is some embodiments, the analytesensing component is present in the sensing membrane in about 40% byweight to about 50% by weight, about 50% by weight to about 60% byweight, about 60% by weight to about 70% by weight, or about 70% byweight to about 80% by weight.

The thickness of the sensing membrane can be from less than about 1 μmto about 10 μm. In some instances, the sensing membrane is less thanabout 2 μm in thickness, where in other applications the copolymer isabout 2 μm to about 3 μm in thickness. In certain applications, thecopolymer is about 2 μm to about 5 μm in thickness, where in otherapplications the copolymer is about 1 μm to about 3 μm or about 4 μm toabout 5 μm in thickness. In some embodiments, the copolymer is about 1μm to about 5 μm in thickness.

In another aspect, the analyte sensor can further include a protectivemembrane adjacent to a surface of the sensing membrane. The protectivemembrane has backbone chains that include:

-   -   first methacrylate-derived units, each having a hydrophilic side        chain;    -   second methacrylate-derived units, each having a hydrophilic        side chain;    -   third methacrylate-derived units; and        hydrophilic crosslinks between the third methacrylate-derived        units in different backbone chains.

Each of the first and second methacrylate-derived units of the backbonescan be independently covalently bound to hydrophilic side chains. Eachof the third methacrylate-derived units is covalently bound through alinker to another third methacrylate-derived unit in a differentbackbone chain. The hydrophilic crosslinks, or groups through which thethird methacrylate-derived units are connected, are discussed in greaterdetail below. Various conformations and compositions of the hydrophilicside chains of the first and second methacrylate-derived units, and thehydrophilic crosslinks of the third methacrylate-derived units can beused to adjust the properties of the crosslinked, hydrophilic copolymer,which include, but are not limited to, hydrophilicity and permeability.

The hydrophilic side chains of the first and second methacrylate-derivedunits can be hydrophilic, and can be water soluble or soluble in awater-miscible solvent, such as an alcohol. The side chains can have oneor more heteroatoms, for example, nitrogen, oxygen or sulfur atoms. Insome embodiments, the side chains have one or more hydroxy groups. Insome embodiments, the side chains of the first and secondmethacrylate-derived units can be the same or substantially the same. Inother embodiments, they are different.

In some embodiments, the hydrophilic side chains of the first and secondmethacrylate-derived units include one or more alkylene oxide units. Thealkylene oxide units can be in the form of a polymer, such aspoly(ethylene glycol), polypropylene glycol), poly(butylene oxide) or amixture thereof, and can be a copolymer including a combination of twoor three different alkylene oxide units. In some embodiments, thepoly(alkylene oxide) of the side chains is a block copolymer includingblocks of two or three different poly(alkylene oxide) polymers. Incertain embodiments, the poly(alkylene oxide) is block copolymer ofpoly(ethylene glycol) and poly(propylene glycol). In other embodiments,the hydrophilic side chain of the second methacrylate-derived unit, andthe crosslinks both include poly(ethylene glycol).

In some embodiments, the first methacrylate-derived units of theprotective membrane can have the structure of formula (II):

where R is a hydrophilic group. In certain embodiments, the hydrophilicgroup includes one or more hydroxy groups, such as an alcohol.

In some embodiments, the first methacrylate-derived units can have thestructure of formula (IIa):

where X is —O—, —NR″— or —S—, y is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,and R¹ is hydrogen, —C₁-C₁₂alkyl, —C₁-C₁₂alkyl-OH, —SiR″₃,—C(O)—C₁-C₁₂alkyl, —C₁-C₁₂alkyl-C(O)OR″, where R″ is hydrogen or—C₁-C₁₂alkyl.

In certain embodiments, the first methacrylate-derived units have thestructure:

In some embodiments, the second methacrylate-derived units of theprotective membrane can have the structure of formula (III):

where Y is —O—, —NR″— or —S—, x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, andR² is hydrogen, —C₁-C₁₂alkyl, —SiR″₃, —C(O)—C₁-C₁₂alkyl,—C₁-C₁₂alkyl-C(O)OR″, where R″ is hydrogen or —C₁-C₁₂alkyl.

In certain embodiments, the second methacrylate-derived units can havethe structure of formula (III) where x is an average value of from about2 to about 250.

In some embodiments, the second methacrylate-derived units can have thestructure of formula (III) where x is such that the poly(ethyleneglycol) has a number average molecular weight (M_(n)) of about 100 toabout 10,000. In certain embodiments, x is selected so that the M_(n) ofthe poly(ethylene glycol) falls within a range in Table 2.

TABLE 2 M_(n) range of poly(ethylene glycol) in the secondmethacrylate-derived units (values are approximate). Low High 100 200200 300 300 400 400 500 500 600 600 700 700 800 800 900 900 1,000 1,0002,000 2,000 3,000 3,000 4,000 4,000 5,000 5,000 6,000 7,000 8,000 8,0009,000 9,000 10,000

In certain embodiments, the second methacrylate-derived units can havethe structure of formula (III), where Y is —O—, R² is methyl and x issuch that the poly(ethylene glycol) has a number average molecularweight (M_(n)) of about 500.

In some embodiments, the presence of the second methacrylate-derivedunits having hydrophilic side chains in the crosslinked, hydrophiliccopolymer of the analyte sensor can form a porous network. The structureof the porous network includes regions within the copolymer that are notoccupied by polymer, these regions are referred to herein as “pores”.The porous network of the crosslinked, hydrophilic copolymer canfacilitate control of the equilibrium between the concentration of theanalyte (e.g., glucose) in the sample solution, and the analyteconcentration in the proximity of the analyte sensor electrode surface.When all of the analyte arriving at the analyte sensor is consumed, themeasured output signal can be linearly proportional to the flow of theanalyte and thus to the concentration of the analyte. However, when theanalyte consumption is limited by the kinetics of chemical orelectrochemical activities in the analyte sensor, the measured outputsignal may no longer be controlled by the flow of analyte and is nolonger linearly proportional to the flow or concentration of theanalyte. In this case, only a fraction of the analyte arriving at theanalyte sensing component is consumed before the sensor becomessaturated, whereupon the measured signal stops increasing, or increasesonly slightly, with an increasing concentration of the analyte. Theporous network can reduce the flow of the analyte to the analyte sensingcomponent so the sensor does not become saturated and can thereforeeffectively enable a wider range of analyte concentrations to bemeasured.

The hydrophilic properties of the hydrophilic side chain of the secondmethacrylate-derived units can be varied to produce desired propertiesof the porous network, such as permeability of the analyte. For example,flow of the analyte into or across the sensor can be dependent on thespecific analyte being monitored, and thus, the porous network can bealtered to obtain properties for monitoring a specific analyte. In someapplications, the hydrophilicity of the porous network can be adjustedby changing the number of alkylene oxide units in the hydrophilic sidechain of the second methacrylate-derived units. Similarly, thehydrophilicity of the porous network can be adjusted by modifying theratio of carbon atoms (i.e., —C—, —CH—, —CH₂— or —CH₃) to alkylene oxideunits in the second methacrylate-derived units.

The crosslinks of the crosslinked, hydrophilic copolymer of theprotective membrane connect the third methacrylate-derived units indifferent backbone chains, and are represented by R^(b) in formula (IV):

where X′ is independently —O—, —NR″— or —S—, and R^(b) is a crosslink,where R″ is hydrogen or —C₁-C₁₂alkyl.

In some embodiments, the crosslinks of the protective membrane can besoluble in water or a water-miscible solvent, such as an alcohol. Thecrosslinks can have one or more heteroatoms, for example, nitrogen,oxygen or sulfur atoms. In some embodiments, the crosslinks have one ormore hydroxy groups.

In some embodiments, the crosslinks of the protective membrane caninclude one or more alkylene oxide units. The alkylene oxide units canbe in the form of a polymer, such as poly(ethylene glycol),poly(propylene glycol), poly(butylene oxide) or a mixture thereof, andcan be a copolymer including a combination of two or three differentalkylene oxide units. In some embodiments, the poly(alkylene oxide) ofthe crosslinks is a block copolymer including blocks of two or threedifferent poly(alkylene oxide) polymers. In certain embodiments, thepoly(alkylene oxide) is a block copolymer of poly(ethylene glycol) andpoly(propylene glycol). In other embodiments, the crosslinks includepoly(ethylene glycol).

In some embodiments, the crosslinks of the protective membrane includeone or more ethylene oxide units. For example, the crosslinks (e.g.,R^(b) in formula IV above) can have the structure of formula (IVa):

where z is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In certain embodiments,the crosslinks of the protective membrane have the structure of formula(IVa) where z is an average value of from about 2 to about 250.

In other embodiments, z in the crosslinks of formula (IVa) is such thatthe number average molecular weight (M_(n)) of the PEG portion (withinthe brackets in formula (IVa)) of the crosslinks is about 100 to about10,000. For example, z can be selected such that the M_(n) of the PEGportion of the crosslinks falls within a range in Table 3:

TABLE 3 M_(n) range of the PEG portion of the crosslinks in theprotective membrane (values are approximate). Low High 100 200 200 300300 400 400 500 500 600 600 700 700 800 800 900 900 1,000 1,000 2,0002,000 3,000 3,000 4,000 4,000 5,000 5,000 6,000 7,000 8,000 8,000 9,0009,000 10,000

In some embodiments, the crosslinks of the protective membrane arederived from di(ethylene glycol) dimethacrylate, i.e., crosslinks offormula (IV) where X′ is —O—, and R^(b) is —CH₂CH₂OCH₂CH₂—, or offormula (IVa) where z is 1.

In some embodiments, the crosslinked copolymer of the protectivemembrane can form a porous network. The structure of the porous networkincludes regions or voids within the copolymer that are not occupied bycopolymer, and are referred to herein as “pores”. The porous network ofthe crosslinked copolymer can facilitate control of the equilibriumbetween the concentration of the analyte (e.g., glucose) in the sample,and the analyte concentration in the proximity of the analyte sensorelectrode surface. When all of the analyte arriving at the analytesensor is consumed, the measured output signal can be linearlyproportional to the flow of the analyte and thus to the concentration ofthe analyte. However, when the analyte consumption is limited by thekinetics of chemical or electrochemical activities in the analytesensor, the measured output signal may no longer be controlled by theflow of analyte and may no longer be linearly proportional to the flowor concentration of the analyte. In this case, only a fraction of theanalyte arriving at the analyte sensing component is consumed before thesensor becomes saturated, whereupon the measured signal stopsincreasing, or increases only slightly, with an increasing concentrationof the analyte. The porous network can reduce the flow of the analyte tothe analyte sensing component so the sensor does not become saturatedand can therefore enable a wider range of analyte concentrations to bemeasured.

The properties of the porous network can be varied to produce desiredproperties, such as permeability of the analyte. For example, flow ofthe analyte into sensing membrane can be dependent on the specificanalyte being monitored, and thus, the porous network can be altered toobtain properties for monitoring a specific analyte. Variation of thesecond methacrylate-derived units can increase or decrease thepermeability of the protective membrane.

The thickness of the crosslinked, hydrophilic copolymer of theprotective membrane can vary depending on the desired properties of theanalyte sensor. The thickness of the copolymer, as measured from theoutward facing surface of sensing membrane to the outward facing surfaceof the copolymer, can play an important role in regulating the flow ofthe analyte to the analyte sensing component. Depending on thecharacteristics of the methacrylate-derived units in the copolymer thetype of analyte sensing component used, and the analyte to be monitored,the thickness of the copolymer can be from less than about 10 μm toabout 30 μm. In some instances, the copolymer is less than 20 μm inthickness, where in other applications the copolymer is about 20 μm toabout 25 μm in thickness. In certain applications, the copolymer isabout 10 μm to about 15 μm in thickness, where in other applications thecopolymer is about 15 lam to about 20 μm or about 25 μm to about 30 μmin thickness. In some embodiments, the copolymer is about 20 μm inthickness.

In some embodiments, the sensing membrane can completely orsubstantially cover one or more surfaces of the electrode to which it isadjacent. In analyte sensors lacking a crosslinked, hydrophiliccopolymer, the sensing membrane can completely or substantially coverone or more surfaces of the electrode, so as to create a barrier ormembrane between the electrode and, when present, an analyte samplebeing monitored or measured. In such embodiments, the sensing membraneis between the electrode and the analyte sample. The sensing membranecan completely cover the electrode to limit the direct contact of theelectrode with the sample.

In analyte sensors having a crosslinked, hydrophilic copolymer, thesensing membrane can act as a barrier or membrane between the electrodeand the crosslinked, hydrophilic copolymer. The sensing membrane cancompletely or substantially cover one or more surfaces of the electrodeto which it is adjacent. The copolymer can be adjacent to, andcompletely or substantially cover, the sensing membrane, so as to createa barrier or membrane between the sensing membrane and, when present,the analyte sample being monitored or measured. In such embodiments, thesensing membrane is between the electrode and the copolymer, and thecopolymer is between the sensing membrane and the analyte sample. Thesensing membrane can completely cover the electrode to limit the directcontact of the electrode with the copolymer and/or the analyte sample,and the copolymer can completely cover the sensing membrane to limit thedirect contact of the analyte sample with the sensing membrane withoutfirst passing through the copolymer.

In some embodiments, the electrode can have an inward facing surface andan outward facing surface, with the outward facing surface completely orsubstantially covered by a sensing membrane. The sensing membrane canalso have an inward facing surface and an outward facing surface. Theoutward facing surface of the electrode can be adjacent to and/or incontact with the inward facing surface of the sensing membrane, and theoutward facing surface of the sensing membrane can be adjacent to and/orin contact with, when present, the analyte sample being monitored ormeasured.

When the analyte sensor comprises a crosslinked, hydrophilic copolymer,the electrode can have an inward facing surface and an outward facingsurface, with the outward facing surface completely or substantiallycovered by a sensing membrane. The sensing membrane can also have aninward facing surface and an outward facing surface. The outward facingsurface of the electrode can be adjacent to and/or in contact with theinward facing surface of the sensing membrane, and the outward facingsurface of the sensing membrane can be completely or substantiallycovered by a crosslinked, hydrophilic copolymer. The copolymer can havean inward facing surface and an outward facing surface. The inwardfacing surface of the copolymer can be adjacent to and/or in contactwith the outward facing surface of the sensing membrane, and the outwardfacing surface of the copolymer can be adjacent to/or in contact with,when present, the analyte sample being monitored or measured.

As used herein, a material that can “completely cover” or a surface thatis “completely covered” refers to greater than about 95% coverage. Insome embodiments, this can refer to greater than about 99% coverage. Asused herein, a material that can “substantially cover” or a surface thatis “substantially covered” refers to greater than about 75% coverage. Insome embodiments, this can refer to great than about 85% coverage, up toabout 95% coverage.

In another aspect, methods for making an analyte sensor are disclosed.The method can involve the formation of a sensing membrane, whichincludes:

-   -   a) forming a sensing mixture including one or more proteins, a        crosslinking agent, and an analyte sensing component, wherein        the proteins have one or more amine functional groups;    -   b) depositing the sensing mixture onto a surface of an        electrode; and    -   c) curing the deposited sensing mixture to provide a sensing        membrane.

In some embodiments of the method for forming the sensing membrane, themixture is formed by combining three separate solutions. The method caninvolve:

-   -   a) forming a first mixture which includes one or more proteins        having one or more amine functional groups;    -   b) forming a second mixture which includes a crosslinking agent;    -   c) forming a third mixture which includes an analyte sensing        component;    -   d) combining the three mixtures to provide the sensing mixture.

In some embodiments, the sensing membrane can be formed on a surface ofan electrode. For example, each component, or a combination of one ormore components, can be individually deposited on a surface of anelectrode to form the deposited sensing mixture. Similarly, when themixture is formed by combining three separate solutions, the solutionscan be combined on a surface of an electrode to form the sensingmixture.

The proteins of the sensing mixture can be selected to provide theproteins of the sensing membrane as discussed herein. In someembodiments, the proteins of the sensing mixture are all the same, orsubstantially the same protein. Two or more proteins can also be used toform the crosslinked sensing membrane. In some embodiments, the proteinsare bovine serum albumin.

The crosslinking agent of the sensing mixture is a chemically reactivespecies that is capable of forming covalent bonds between the proteinsof the sensing membrane, and/or covalent bonds between the analytesensing component and one or more proteins and/or another analytesensing component in the sensing membrane. In some embodiments, thecrosslinking agent can form carbon-nitrogen double bonds between thenitrogen atoms of amine groups on the proteins and/or analyte sensingcomponent and carbon atoms in the crosslinking agent. In someembodiments, the crosslinking agent can be a di-carbonyl compound. Forexample, the crosslinking agent can have the structure of formula (V):

where R^(c) and R′ are selected to provide the crosslinks of the sensingmembrane described herein. In some embodiments, R^(c) is C₀-C₄alkyl or ahydrophilic group, and R′ is independently hydrogen, chloro,—C₁-C₁₂alkyl or N-hydroxysuccinimide. In some embodiments, thecrosslinking agent can be oxaldehyde, malonaldehyde, succinaldehyde,glutaraldehyde or adipaldehyde. In other embodiments, the hydrophilicgroup can be selected to provide the crosslinks of the sensing membranedescribed herein.

The analyte sensing component can be selected based on the analytedesired to be monitored. For example, to monitor physiologicalcholesterol levels, cholesterol oxidase can be used, and to monitorlactate levels lactate oxidase can be used. To monitor glucose levels,the analyte sensing component can include glucose oxidase or glucosedehydrogenase (GDH).

The analyte sensing component can be present when the proteins in thedeposited sensing membrane mixture are crosslinked, such thatcrosslinking results in the formation of a crosslinked network in whichthe analyte sensing component is embedded. The embedded analyte sensingcomponent is immobilized and can be used to monitor a correspondinganalyte of interest.

In some embodiments, the method further includes the formation of aprotective membrane, which includes:

-   -   a) forming a copolymer mixture including an initiator, a first        methacrylate monomer having a hydrophilic side chain, a        dimethacrylate monomer, and a second methacrylate monomer having        a hydrophilic side chain;    -   b) depositing the copolymer mixture onto a surface of the        sensing membrane; and    -   c) subjecting the deposited copolymer mixture to conditions        sufficient to initiate polymerization (i.e., curing).

In some embodiments of the method, the copolymer mixture is formed bycombining separate solutions of the copolymer precursors. The method caninvolve:

-   -   a) forming a first mixture which includes a dimethacrylate        monomer, an initiator, and first methacrylate monomer having a        hydrophilic side chain;    -   b) forming a second mixture which includes a dimethacrylate        monomer, an initiator, and second methacrylate monomer having a        hydrophilic side chain; and    -   d) combining the mixtures to provide the copolymer mixture.

In some embodiments of the method for forming the protective membrane,the initiator and/or the dimethacrylate monomer can be present in any ofthe mixtures. For example, the first or second mixture can include aninitiator and/or a dimethacrylate monomer. In other embodiments of themethod for forming the protective membrane, the third mixture is notpresent and the dimethacrylate monomer is present in the first and/orsecond mixture.

The first and second methacrylate monomers of the copolymer mixtureinclude hydrophilic side chains that can have one or more heteroatoms.The hydrophilic side chains can include one or more alkylene oxide unitsto form the crosslinked, hydrophilic copolymer of the analyte sensor asdescribed herein.

The first methacrylate monomer of the copolymer mixture can be selectedto provide the first methacrylate-derived units of the crosslinked,hydrophilic copolymer as described herein. In some embodiments of themethod, the first methacrylate monomer has the structure of formula(VI):

where R is a hydrophilic group. In certain embodiments of the method,the hydrophilic group includes one or more hydroxy groups, such as analcohol.

In some embodiments of the method, the first methacrylate monomer hasthe structure of formula (VIa):

where X, y, R¹, and R″ are selected to provide the firstmethacrylate-derived monomeric unit of the crosslinked, hydrophiliccopolymer described herein.

In certain embodiments of the method, the first methacrylate monomer hasthe structure:

The second methacrylate monomer of the copolymer mixture can be selectedto provide the second methacrylate-derived units of the crosslinked,hydrophilic copolymer as described herein. In some embodiments of themethod, the second methacrylate monomer has the structure of formula(VII):

where Y, x, R² and R″ are selected to provide the secondmethacrylate-derived monomeric unit of the crosslinked, hydrophiliccopolymer described herein.

In some embodiments of the method, the second methacrylate monomer hasthe structure of formula (VII) where x is selected to provide secondmethacrylate-derived monomeric units of the crosslinked, hydrophiliccopolymer described herein where the poly(ethylene glycol) has a numberaverage molecular weight (M_(n)) of about 100 to about 10,000. Incertain embodiments, x is selected to provide secondmethacrylate-derived monomeric units where the M_(n) of thepoly(ethylene glycol) falls within a range in Table 2.

In certain embodiments of the method, the second methacrylate monomerhas the structure of formula (VII), where Y is —O—, R² is methyl and xis such that the poly(ethylene glycol) has a number average molecularweight (M_(n)) of about 500.

The dimethacrylate monomer of the copolymer mixture is a molecule havingtwo terminal methacrylate groups tethered by a hydrophilic linker. Thehydrophilic linker is selected to provide the crosslinks between thirdmethacrylate-derived units in different backbone chains of thecrosslinked, hydrophilic copolymer described herein. In embodimentswhere the mixture is formed from the combination of two or moresolutions each having a dimethacrylate monomer, the dimethacrylatemonomers can be the same, or in some instances, can be different.

The extent of crosslinking in crosslinked, hydrophilic copolymer of theprotective membrane can be controlled by adjusting the amount ofdimethacrylate monomer in the mixture. In some embodiments, thedimethacrylate monomer can be about 1% to about 15% of the mixture. Inother examples, the amount is about 1% to about 5%, or about 5% to about10%, or about 10% to about 15%. In some embodiments, the amount is about1%. In some instances, both the first and second mixtures include about1% of the dimethacrylate monomer.

The dimethacrylate monomer of the copolymer mixture can be selected toprovide the crosslinks of the crosslinked, hydrophilic copolymer asdescribed herein. In some embodiments of the method, the dimethacrylatemonomer includes one or more alkylene oxide units to provide thecrosslinks of the crosslinked, hydrophilic copolymer as describedherein. In some embodiments, the dimethacrylate monomer includespoly(ethylene glycol) (PEG). For example, the dimethacrylate monomer canhave the structure of formula (VIII):

where z is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In certain embodiments of the method, the dimethacrylate monomer canhave the structure of formula (VIII) where z is an average value of fromabout 2 to about 250.

In other embodiments of the method, the dimethacrylate monomer can havethe structure of formula (VIII) where z is such that the number averagemolecular weight (M_(n)) of the PEG portion of the dimethacrylatemonomer is about 100 to about 10,000. For example, w can be selectedsuch that the M_(n) of the PEG portion of the dimethacrylate monomerfalls within a range in Table 3. In some embodiments, the dimethacrylatemonomer is di(ethylene glycol) dimethacrylate.

The sensing and copolymer mixtures of the method can be formed in anaqueous medium, alcoholic medium, or mixture thereof. The aqueous mediumcan include a buffered aqueous solution, such as, for example, asolution containing citric acid, acetic acid, borate, carbonate,bicarbonate, 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES),3-{[tris(hydroxymethyl)methyl]amino-}-propanesulfonic acid (TAPS),N,N-bis(2-hydroxyethyl)glycine (Bicine), tris(hydroxymethyl)methylamine(Tris), N-tris(hydroxymethyl)methylglycine (Tricine),3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid(TAPSO), 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES),3-(N-morpholino)propanesulfonic acid (MOPS),piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), dimethylarsinic acid(Cacodylate), saline sodium citrate (SSC),2-(N-morpholino)ethanesulfonic acid (MES), 2(R)-2-(methylamino)succinicacid, or phosphate buffered saline (PBS). In some embodiments, themixture can be formed in a mixture of a buffered aqueous solution andethanol.

In some embodiments of the method, the mixtures and solutions of themethod can be formed with approximately the same concentration ofcomponent (e.g., protein, analyte sensing component, first methacrylatemonomer, etc.). The percentage of each component can then be varied byadjusting the amounts of each individual mixture used to form themixture used for forming the sensing or protective membranes. In someinstances, the percentage of analyte sensing component in the sensingmembrane mixture, can be about 40% by weight to about 80% by weight, theproteins can be about 15% by weight to about 50%, and the crosslinkingagent can be about 1% by weight to about 10% by weight. In certainexamples, the percentage of analyte sensing component in the sensingmembrane mixture, can be about 60% by weight to about 70% by weight, theproteins can be about 25% by weight to about 35% by weight, and thecrosslinking agent can be about 1% by weight to about 5% by weight. Inthe protective membrane mixture, the percentage of first methacrylatemonomer can be about 20% by weight to about 60% by weight, thepercentage of second methacrylate monomer can be about 10% by weight toabout 40% by weight, and the percentage of dimethacrylate monomer can beabout 0.1% by weight to about 5% by weight. All percentages are given asa percentage of the cumulative amount of analyte sensing component,first methacrylate monomer and second methacrylate monomer. In certainexamples, the percentage of analyte sensing component is about 40% byweight, the amount of first methacrylate monomer is about 35% by weightto about 40% by weight, and the amount of second methacrylate monomer isabout 20% by weight to about 25% by weight. In certain embodiments ofthe method, the mixture can be thoroughly mixed, optionally with astirrer or shaker, before being deposited.

The ratio of the components in each mixture can vary depending on thedesired properties of the resulting analyte sensor. For example,adjusting the amount of the second methacrylate monomer having ahydrophilic side chain can alter the porous network of the protectivemembrane. Controlling the properties of the porous network can allow forthe tuning of the permeability of the analyte sensor. Similar tunabilitycan also be accomplished by adjusting the amount of the sensing mixturedeposited on the electrode, and/or adjusting the amount of the secondmethacrylate monomer combined with the first methacrylate monomer.

Depositing the sensing mixture onto a surface of an electrode, or thecopolymer mixture onto a surface of the sensing membrane can beaccomplished by a number of methods. For example, the depositing can beperformed manually with a micro-syringe, or by automated fabricationprocesses with nano jet dispensing equipment.

In some embodiments of the methods, the amount of the sensing orcopolymer mixture deposited is selected to provide the desired thicknessof the sensing or protective membrane of the analyte sensor,respectively. In some embodiments, the amount deposited on the electrodeis about 50 nL/mm² to about 500 nL/mm². In other examples, the amount isabout 50 μm to about 150 μm, or about 150 μm to about 300 μm, or about300 μm to about 500 μm in thickness. In some embodiments, the amount isabout 100 nL/mm². In some instances, depositing about 100 nL/mm² of thesensing or copolymer mixture provides a sensing membrane or protectivemembrane, respectively, that is about 20 μm in thickness.

Conditions suitable to initiate polymerization (i.e., curing) can beselected based on the characteristics of the components beingpolymerized, and as so not to degrade the analyte sensing component. Inembodiments where the analyte sensing component is an enzyme, thetemperature and pH of the method can be selected to preserve theactivity of the enzyme. In certain embodiments the initiator isactivated with ultraviolet (UV) light. For example, when2,2-diemthoxy-2-phenylacetophenone is used as an initiator, curing canbe performed with UV light. In other instances, curing is performed byair-drying at ambient or elevated temperature. In embodiments where themixture is formed from the combination of two or more solutions eachhaving an initiator, the initiators can be the same, or in someinstances, can be different.

Although the crosslinked, hydrophilic copolymers in the above examplescomprise methacrylate groups, there are a number of ethylenicallyunsaturated groups known in the art to be capable of undergoingpolymerization. Ethylenically unsaturated monomers and macromers may beeither acrylic- or vinyl-containing. Vinyl-containing monomers containthe vinyl grouping (CH₂═CH—), and are generally highly reactive.Acrylic-containing monomers are represented by the formula:

Examples of suitable polymerizable groups may include acrylic-,ethacrylic-, itaconic-, styryl-, acrylamido-, methacrylamido- andvinyl-containing groups such as the allyl group.

In addition to the above disclosed methods of forming crosslinked,hydrophilic copolymers by the polymerization of ethylenicallyunsaturated monomers and macromonomers, additional chemistries will beknown to one or ordinary skill in the art to from such copolymers. As anexample, epoxy chemistry, in which multifunctional amines andmultifunctional epoxy compounds are mixed together and cured, can beused to form crosslinked, hydrophilic copolymers. Additionally, urethanechemistry may be used, in which multifunctional isocyanates are mixedwith multifunctional alcohols and cured to provide crosslinked,hydrophilic copolymers. Other chemistries for the formation ofcrosslinked, hydrophilic copolymers exist, and will be well known tothose of ordinary skill in the art.

It should be understood that arrangements described herein are forpurposes of example only. As such, those skilled in the art willappreciate that other arrangements and other elements (e.g., machines,interfaces, functions, orders, and groupings of functions, etc.) can beused instead, and some elements can be omitted altogether according tothe desired results. Further, many of the elements that are describedare functional entities that can be implemented as discrete ordistributed components or in conjunction with other components, in anysuitable combination and location.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims, along with the fullscope of equivalents to which such claims are entitled. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Further, some embodiments of the system may include privacy controlswhich may be automatically implemented or controlled by the wearer ofthe device. For example, where a wearer's collected physiologicalparameter data and health state data are uploaded to a cloud computingnetwork for trend analysis by a clinician, the data may be treated inone or more ways before it is stored or used, so that personallyidentifiable information is removed. For example, a user's identity maybe treated so that no personally identifiable information can bedetermined for the user, or a user's geographic location may begeneralized where location information is obtained (such as to a city,ZIP code, or state level), so that a particular location of a usercannot be determined.

Additionally or alternatively, wearers of a device may be provided withan opportunity to control whether or how the device collects informationabout the wearer (e.g., information about a user's medical history,social actions or activities, profession, a user's preferences, or auser's current location), or to control how such information may beused. Thus, the wearer may have control over how information iscollected about him or her and used by a clinician or physician or otheruser of the data. For example, a wearer may elect that data, such ashealth state and physiological parameters, collected from his or herdevice may only be used for generating an individual baseline andrecommendations in response to collection and comparison of his or herown data and may not be used in generating a population baseline or foruse in population correlation studies.

EXAMPLES Example 1 Formation of a Two-Layer Analyte Sensor

Formation of the sensing membrane:

A solution of glucose oxidase (10.0 mg) and BSA (Albumin from bovineserum, 4.0 mg) in 200 uL PBS buffer was formed. To this mixture wasadded 12.0 uL glutaraldehyde (50% solution in water), and the resultingmixture was mixed. The mixture was deposited (120 mL) onto a sensor areaof 1×3 mm, and air dried overnight at ambient temperature and pressure.

Formation of the protective membrane:

Two solutions (A and B) were prepared:

-   -   A) 2-hydroxyethyl methacrylate monomer solution containing 1% by        weight di(ethylene glycol) dimethacrylate and 1% by weight        2,2-dimethoxy-2-phenylacetophenone.    -   B) poly(ethylene glycol) methyl ether methacrylate (average Mn        500, Aldrich product #447943) monomer solution containing 1% by        weight di(ethylene glycol) dimethacrylate and 1% by weight        2,2-dimethoxy-2-phenylacetophenone.        The two solutions were combined with PBS buffer in a ratio of        0.225:0.175:0.200 A:B:PBS and thoroughly mixed with a vortex        shaker. The resulting mixture was deposited (100 nL/mm²) onto a        surface of the sensing membrane, and the deposited mixture was        UV-cured for 5 minutes at 365 nm under nitrogen with an EC-500        light exposure chamber (Electro-Lite Corp). The resulting cured        crosslinked, copolymer protective membrane had a thickness of        about 20 μm.

Example 2 Analyte Sensor Performance in a Glucose Solution

Six analyte sensors made according to Example 1 were tested atconcentrations of glucose in phosphate buffered saline (PBS) rangingfrom 50 μM to 1000 μm. The sensors were submerged in PBS and the glucoseconcentration was increased every 10-15 minutes. The current generatedat the electrode of each sensor was measured using a potentiostat (FIG.1). A linear relationship between current and glucose concentration wasobserved (FIG. 2).

1. An analyte sensor comprising: a sensing membrane comprising: acrosslinked network comprising one or more proteins and crosslinks,wherein the proteins are crosslinked through carbon-nitrogen doublebonds between amine group nitrogen atoms on the proteins and carbonatoms on the crosslinks; and an analyte sensing component embedded inthe crosslinked network.
 2. The analyte sensor of claim 1, wherein inthe crosslinks are C₂-C₆alkyl.
 3. The analyte sensor of claim 1, whereinthe crosslinks comprise one or more alkylene oxide unit.
 4. The analytesensor of claim 1, wherein the proteins are bovine serum albumin.
 5. Theanalyte sensor of claim 1, wherein the analyte sensing componentcomprises glucose oxidase.
 6. The analyte sensor of claim 1, wherein thesensing membrane is adjacent to a surface of an electrode.
 7. Theanalyte sensor of claim 1, further comprising a protective membrane,wherein the protective membrane is adjacent to a surface of the sensingmembrane.
 8. The analyte sensor of claim 7, where in the protectivemembrane comprises a crosslinked, hydrophilic copolymer comprising:backbone chains comprising: first methacrylate-derived units, eachhaving a hydrophilic side chain; second methacrylate-derived units, eachhaving a hydrophilic side chain; third methacrylate-derived units; andhydrophilic crosslinks between the third methacrylate-derived units indifferent backbone chains.
 9. The analyte sensor of claim 3, wherein thesensing membrane is between the electrode and the crosslinked,hydrophilic copolymer.
 10. The analyte sensor of claim 8, wherein thefirst methacrylate-derived units have the structure of formula (IIa):

wherein X is —O—, —NR″— or —S—; y is 0-10; and R¹ is hydrogen,—C₁-C₁₂alkyl, —C₁-C₁₂alkyl-OH, —SiR″₃, —C(O)—C₁-C₁₂alkyl,—C₁-C₁₂alkyl-C(O)OR″, wherein R″ is —C₁-C₁₂alkyl.
 11. The analyte sensorof claim 8, wherein the first methacrylate-derived units have thestructure:


12. The analyte sensor of claim 8, wherein the hydrophilic side chain ofthe second methacrylate-derived units comprises one or more alkyleneoxide unit.
 13. The analyte sensor of claim 12, wherein the secondmethacrylate-derived units have the structure of formula (III):

wherein Y is —O—, —NR″— or —S—; R² is hydrogen, —C₁-C₁₂alkyl, —SiR″₃,—C(O)—C₁-C₁₂alkyl, —C₁-C₁₂alkyl-C(O)OR″, where R″ is hydrogen or—C₁-C₁₂alkyl; and x is 1-10.
 14. The analyte sensor of claim 12, whereinthe second methacrylate-derived units have the structure of formula:(III):

wherein Y is —O—, —NR″— or —S—; R² is hydrogen, —C₁-C₁₂alkyl, —SiR″₃,—C(O)—C₁-C₁₂alkyl, —C₁-C₁₂alkyl-C(O)OR″, where R″ is hydrogen or—C₁-C₁₂alkyl; and x is an average value of from 2 to about
 250. 15. Theanalyte sensor of claim 7, wherein the protective membrane has athickness of about 20 μm.
 16. The analyte sensor of claim 1, wherein theproteins are BSA; the crosslinks are C₂-C₆alkyl; the analyte sensingcomponent comprises glucose oxidase;


17. The analyte sensor of claim 8, wherein the firstmethacrylate-derived units are derived from 2-hydroxyethylmethacrylate;the second methacrylate-derived units have the structure of formula(III):

wherein x is an average value of from about 10 to about 15; and thehydrophilic crosslinks have the structure of formula (IVa):

wherein z is
 1. 18. The analyte sensor of claim 8, wherein the proteinsare BSA; the crosslinks are C₅alkyl; the analyte sensing componentcomprises glucose oxidase; the first methacrylate-derived units arederived from 2-hydroxyethylmethacrylate; the second methacrylate-derivedunits have the structure of formula (III):

wherein x is an average value of from about 10 to about 15; and thehydrophilic crosslinks have the structure of formula (IVa):

wherein z is
 1. 19. A method for manufacturing an analyte sensor, themethod comprising: forming a sensing mixture comprising one or moreproteins, a crosslinking agent, and an analyte sensing component,wherein the proteins have one or more amine functional groups;depositing the sensing mixture onto a surface of an electrode; curingthe deposited sensing mixture to provide a sensing membrane.
 20. Themethod of claim 19, further comprising: forming a copolymer mixturecomprising an initiator, a first methacrylate monomer having ahydrophilic side chain, a dimethacrylate monomer, and a secondmethacrylate monomer having a hydrophilic side chain; depositing thecopolymer mixture onto a surface of the sensing membrane; and curing thedeposited copolymer mixture to provide a protective membrane.